Freeze-dried composition containing anti-HER2 drug conjugate, freeze-dried preparation and preparation method therefor and use thereof

Abstract

The present invention provides a freeze-dried composition containing anti-HER2 drug conjugate, which comprises ARX788 and one or more of pharmaceutically acceptable lyoprotectant, buffer salts and surfactants, wherein the pH value of the freeze-dried composition is from 5.7 to 6.3. The present invention also provides a freeze-dried preparation containing anti-HER2 drug conjugate and the preparation method thereof. The present invention also provides use of the freeze-dried composition and the freeze-dried preparation. The freeze-dried composition and the freeze-dried preparation provided by the present invention have a very excellent stability, as well as good appearance, short reconstitution time and low moisture content. The product quality is safe and reliable, and the production efficiency is high. The preparation methods of the freeze-dried composition and the freeze-dried preparation provided by the present invention are simple and have low-cost, and they can be used for treating various cancers, and have broad application prospects.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a 371 U.S. National Phase of PCT International Application No. PCT / CN2021 / 107402 filed on Jul. 20, 2021. The content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present invention relates to the field of biopharmaceutical technology, in particular to a freeze-dried composition containing anti-HER2 drug conjugate, a freeze-dried preparation prepared from the freeze-dried composition, and a preparation method thereof and use thereof.BACKGROUND

[0003] Human epidermal growth factor receptor 2 (HER2 / ErbB2) is a member of the epithelial growth factor receptor family, which is a transmembrane tyrosine kinase. The amplification of HER2 gene exists in about 30% of the primary human breast cancers, and sometimes it is also found in many other solid tumors. The amplification of HER2 gene usually leads to overexpression, resulting in up to one million HER2 protein molecules on the surface of each cancer cell. Increased HER2 protein expression and HER2 gene amplification are associated with poor clinical prognosis, including shortened relapse-free survivals and overall survivals (OSs) of breast cancer and gastric cancer, and shortened overall survivals of lung cancer, ovarian cancer, colon cancer and pancreatic cancer. Several therapeutic agents, antibodies and antibody-drug conjugates (ADCs, including T-DM1) targeting HER2 have been proved to have clinical anticancer activity.

[0004] A pharmaceutical must be marketed in the form of final preparation, and the selection of dosage form, the types and dosages of excipients, and the preparation process all have an impact on the stability of the product. A qualified product should have stable and controllable quality within its validity period, and be convenient for transportation, storage and clinical use. Pharmaceutical stability is one of the important indicators for evaluating pharmaceutical effectiveness and safety. Stability research is the main basis for determining the storage condition and shelf life of a pharmaceutical, which can also provide scientific basis for the pharmaceutical production, packaging, transportation condition and so on. For a pharmaceutical, especially a biological product, the maintenance of molecular configuration and biological activity of the active ingredient depends on various covalent and non-covalent forces, and the maintenance of these forces are closely related to the microenvironment they are in, such as temperature, light, ion concentration, and mechanical shear force.

[0005] ADC is a highly effective and specific pharmaceutical used to treat cancers and other diseases, in which the antibody part specifically binds to the antigen thereof on the target cells, allowing the drug to exert its cytotoxicity or other therapeutic effects on the target cells. However, like other protein pharmaceuticals, the antibody is prone to degradation, such as through oxidation, deamidation and fragmentation, as well as to form particles and aggregates. The purpose of developing an ADC formulation is to ensure stable administration to patients and reliable quality. However, compared with naked antibody, ADC has a more complex structure and has more unstable factors. The numerous formulation characteristics which are exhibited during the development process of a biological preparation may affect the safety, effectiveness and quality controllability of the product. Therefore, ADC poses a challenge to the preparation used for therapeutic purpose. In order to provide a stable ADC pharmaceutical during transportation and storage, careful selections of the carrier, excipient, and / or stabilizer in the pharmaceutical preparation are necessary.

[0006] The ADC quality attribute includes the aspect of free small molecule drug, and the aspect of naked antibody, as well as the aspect of antibody drug conjugate. The content of free payload may affect the safety of the ADC, while the naked antibody itself has inherent critical quality attribute of an antibody, such as chemical instability, like aggregation, fragmentation, charge heterogeneity, degradation, oxidation and so on, which may affect a variety of clinical effects such as biological activity, immunogenicity, drug efficacy and safety. The unique quality attribute of an ADC, such as chemical stability and drug-antibody ratio of the conjugate, may affect the efficacy, drug metabolism and safety of the ADC.SUMMARY

[0007] Based on the particularity of an ADC preparation, in order to provide a novel and stable ADC preparation, one object of the present invention is to provide a freeze-dried composition containing anti-HER2 drug conjugate.

[0008] Another object of the present invention is to provide a freeze-dried preparation containing anti-HER2 drug conjugate and a preparation method thereof.

[0009] Further object of the present invention is to provide use of the freeze-dried composition and the freeze-dried preparation.

[0010] The freeze-dried composition containing anti-HER2 drug conjugate provided by the present invention comprises ARX788 and one or more of pharmaceutically acceptable lyoprotectant, buffer salt and surfactant, wherein the pH value of the freeze-dried composition is from 5.7 to 6.3.

[0011] In some preferred embodiments, the pH value of the freeze-dried composition may be from 6.0 to 6.3, and in some most preferred embodiments, the pH value of the freeze-dried composition may be 6.0.

[0012] ARX788 described in the present invention is an anti-HER2 ADC, also known as anti-HER2 monoclonal antibody AS269 conjugate (the structural formula thereof is shown in FIG. 1). In ARX788, the anti-HER2 antibody is obtained by using codon expansion technology to mutate the amino acid at 121st position of each heavy chain of trastuzumab into a p-acetyl phenylalanine residue, which is then subjected to oximation reaction with a small molecule toxin containing a hydroxylamine group for fixed coupling to give the product with a main drug-to-antibody ratio of 1:2. Chinese patent CN 201280036296.1 provides a detailed description of this.

[0013] The pharmaceutically acceptable lyoprotectant, buffer salt and surfactant described in the present invention may be any type commonly used in pharmaceutical field, especially those commonly used in ADC preparations. In some preferred embodiments, the buffer salt includes but is not limited to acetate, phosphate, citrate, histidine salt, succinate, 2-(N-morpholinyl) ethanesulfonic acid, edetate disodium, etc: the surfactant includes but is not limited to polysorbate 20, polysorbate 80, etc: the lyoprotectant includes but is not limited to sucrose, trehalose, etc.

[0014] In some preferred embodiments, the freeze-dried composition described in the present invention comprises 10 mg / mL to 30 mg / mL of ARX788, 5 mM to 10 mM of histidine, 2.5 wt. % to 8 wt. % trehalose and 0.01 wt. % to 0.05 wt. % of polysorbate 80.

[0015] In some more preferred embodiments, the freeze-dried composition described in the present invention comprises 20 mg / mL of ARX788, 5 mM of histidine, 6 wt. % trehalose and 0.02 wt. % of polysorbate 80, and the pH of the freeze-dried composition is 6.0.

[0016] The present invention also provides a freeze-dried preparation containing anti-HER2 drug conjugate, which is prepared from the freeze-dried composition described in any one of the above technical solutions.

[0017] The present invention also provides a preparation method for the freeze-dried preparation, comprising the following steps:

[0018] S1: prefreezing the freeze-dried composition described in any one of the above technical solutions at −50° C. to −40° C. for 3 hours to 6 hours;

[0019] S2: heating up the freeze-dried composition after treated in step S1 to −25° C. to −20° C. and holding at this temperature and a vacuum degree of 10 Pa to 15 Pa for 30 hours to 50 hours; and

[0020] S3: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. and holding at this temperature and a vacuum degree of 10 Pa to 15 Pa for 5 hours to 10 hours.

[0021] In some preferred embodiments, in the preparation method provided by the present invention, the step S1 comprises: cooling down the freeze-dried composition described in any one of the above technical solutions to 2° C. to 5° C. within 0.5 hour to 1.5 hours and holding at the same temperature for 0.5 hour to 1.5 hours, then cooling down to −50° C. to −40° C. within 3 hours to 5 hours and holding at the same temperature for 3 hours to 6 hours. In some more preferred embodiments, the step S1 comprises: cooling down the freeze-dried composition described in any one of the above technical solutions to 4° C. within 1 hour and holding at the same temperature for 1 hour, then cooling down to −45° C. within 3.5 hours and holding at the same temperature for 5 hours.

[0022] In some preferred embodiments, in the preparation method provided by the present invention, the step S2 comprises: heating up the freeze-dried composition after treated in step S1 to −23° C. to −20° C. within 0.2 hour to 0.8 hour and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 35 hours to 45 hours. In some more preferred embodiments, the step S2 comprises: heating up the freeze-dried composition after treated in step S1 to −23° C. to −20° C. within 0.5 hour and holding at the same temperature and a vacuum degree of 13 Pa for 40 hours.

[0023] In some preferred embodiments, in the preparation method provided by the present invention, the step S3 comprises: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. within 2 hours to 8 hours and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 5 hours to 10 hours. In some more preferred embodiments, the step S3 comprises: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. within 5 hours and holding at the same temperature and a vacuum degree of 13 Pa for 8 hours.

[0024] The present invention also provides use of the freeze-dried composition described in any one of the above technical solutions or the freeze-dried preparation described in any one of the above technical solutions for the preparation of a medicament for treating a cancer.

[0025] In the use of the present invention, the cancer includes but is not limited to breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, urothelial cancer, biliary tract cancer, liver cancer, brain cancer, esophageal cancer, endometrial cancer, uterine cancer, kidney cancer, bladder cancer, thyroid cancer, salivary gland cancer or pancreatic cancer.

[0026] The freeze-dried composition and the freeze-dried preparation provided by the present invention are specifically designed for the novel ADC drug ARX788. By setting specific pH value, composition formula and even freeze-drying process, the drop of the small molecule drug and the decline of CEX main peak can be effectively reduced. The resulting freeze-dried product has very excellent stability and can maintain good stability and biological activity for a long time even at high temperatures (such as 40° C.). Moreover, the freeze-dried product also has good appearance, short reconstitution time (within one minute, even up to within 30 seconds) and low moisture content. The product quality is safe and reliable, and the production efficiency is high. The preparation methods of the freeze-dried composition and the freeze-dried preparation provided by the present invention are simple and have low-cost, and they can be used for treating various cancers, and have broad application prospects.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0027] FIG. 1 shows the structure diagram of ADC drug ARX788, wherein one antibody protein is coupled with two small molecule compounds.

[0028] FIG. 2A and FIG. 2B respectively show the charts of CEX changing trend and potency changing trend under the investigation with high temperature condition in Example 5.DETAILED DESCRIPTIONS

[0029] The technical solutions of the present invention will be further described in detail below in combined with specific examples.

[0030] The ADC drug ARX788 and ADC reference substance (RS) used in the examples are provided by Zhejiang Novocodex Biopharmaceuticals Co., Ltd. Other reagents or raw materials are commercially available if not particularly indicated.

[0031] The test items and test conditions used in the examples are described as follows:

[0032] Appearance: measured by visual inspection with clarity detector.

[0033] Protein concentration: measured by Cary 100 UV-VIS spectrophotometer.

[0034] pH value: instrument: Mettler InLab® Micro Pro pH meter.Cation-Exchange Chromatography CEX-HPLC:

[0035] ChromatographicDionex BioLC ProPac WCX-10, 4 × 250 mm 10 μmcolumnDetector wavelength280 nmTemperature of40° C.temperature-controlled ovenAuto-Sampler5° C.temperatureFlow rate1.0 mL / minInjection volume100 μLMobile phase A20 mM NaH2PO4•2H2O, pH 6.5 ± 0.1Mobile phase B20 mM NaH2PO4•2H2O, 200 m M NaCl, pH6.5 ± 0.1Gradient elutionTime (min)A %B %09010590107560408060408201008501008790101009010Residual Amount of AS269 (Small Molecule Toxin):

[0036] ChromatographicWaters XBridge Shield RP 18, 3.5 μm, 1*100 mmcolumnDetector wavelength214 nmTemperature of40° C.temperature-controlled ovenAuto-Sampler5° C.temperatureFlow rate0.8 mL / minInjection volume100 μLMobile phase A0.1% TFA / waterMobile phase B0.1% TFA / ACNGradient elutionTime (min)A %B %080209604012208012.18020158020Cell-Based Potency Test:

[0037] The cell-based potency of the freeze-dried sample relative to RS was tested by using BT-474 cell line (ATCC, article No. HTB-20).

[0038] The specific process was as follows: BT-474 cells were cultured to obtain a sufficient quantity with ATCC Hybrid Care Medium (ATCC, article No. 46-X) containing 10% fetal bovine serum (Gibco, article No. 10099141) under the conditions of 37° C. and 5% carbon dioxide. 1.5×104 of BT-474 cells were inoculated into each well in a 96-well cell culture plate (Corning, article No. 3599), with an amount of 80 μL / well. The freeze-dried sample and RS were subjected to gradient dilution from 55.8 μg / mL to 0.2 μg / mL, with a total of 10 concentrations and 2 parallel wells for each dilution. The diluted freeze-dried sample and RS were transferred to the culture plate inoculated with BT-474 cells, with an amount of 10 μL per well, and the plate was cultured under the conditions of 37° C. and 5% carbon dioxide. 10 μL of CCK-8 (Beyotime, article No. C0043) test reagent was added into each well at day 5 for coloration under the conditions of 37° C. and 5% carbon dioxide for 6 hours. The optical density values were read by a microplate reader at 450 nm and 620 nm wavelengths. EC50 of the freeze-dried sample and RS were calculated by using SoftMax Pro software, and the relative cell-based potency of the freeze-dried sample was calculated by using the following formula: relative cell-based potency (%)=(EC50 RS) / (EC50 freeze-dried sample)×100%.Binding Potency Test:

[0039] ELISA was used to test the binding potency of the freeze-dried sample relative to RS.

[0040] The specific process was as follows: HER2 antigen (R&D, article No. 1129-ER-050) was added into a 96-well plate (Corning, article No. 9018) with an amount of 50 μL / well to be coated overnight at 2° C. to 8° C. Blocking solution (Fisher Scientific, article No. 37528) was added with an amount of 200 μL / well, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. In the 96-well plate, the freeze-dried sample and RS were subjected to gradient dilution from 10 μg / mL to 0.00017 μg / mL by using the blocking solution, with a total of 11 concentrations and 2 parallel wells for each dilution, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. Second antibody (Abcam, article No. ab79115) was added with an amount of 50 μL / well, and the plate was shaken at 200±10 rpm for 60 minutes at room temperature. TMB coloring solution (Beyotime, article No. P0209) was added with an amount of 50 μL / well, and the plate was shaken at 200±10 rpm for 10 minutes at room temperature. 5% sulfuric acid was added with an amount of 50 μL / well for termination, and the light absorbance values were read at 450 nm and 620 nm respectively. EC50 of the freeze-dried sample and RS were calculated by using SoftMax Pro software, and the relative binding potency of the freeze-dried sample was calculated by using the following formula: relative binding potency (%)=(EC50 RS) / (EC50 freeze-dried sample)×100%.

[0041] Unless otherwise specified, the percentages used in the examples are all mass percentages.Example 1 Stability Study Under Different pH Values

[0042] Formulations 1 to 10 with different pH values were designed (see Table 1).

[0043] TABLE 1Formulations with different pH valuesAnti-HER2PolysorbateFormulationADCBufferBuffer saltTrehalose80pHnumberconcentrationsalt typeconcentrationconcentrationconcentrationvalueFormulation 110 mg / mlHistidine5 mM50 mg / ml0.2 mg / ml5.4Formulation 2salt5.7Formulation 36.0Formulation 46.3Formulation 57.2Formulation 6Acetate4.0Formulation 74.5Formulation 85.0Formulation 95.5Formulation 106.0Samples Preparation:

[0044] (1) 5 mM acetate buffer solution and histidine salt buffer solution were prepared respectively, and adjusted to the target pH values with hydrochloric acid or sodium hydroxide.

[0045] (2) Excipient stock solutions were prepared by using the buffer salt solutions, with the polysorbate 80 concentration of 14 mg / ml (70 times of the target concentration) and the trehalose concentration of 500 mg / ml (10 times of the target concentration) respectively.

[0046] (3) 10 ADC solutions were taken and added into pre-treated ultrafiltration centrifuge tubes (model: 30 k, 15 ml) respectively. The tubes were centrifuged for 20 minutes, then added with the buffer salt solutions and centrifuged for another 20 minutes for solution replacement. The solution replacement and concentration operations were repeated four times to give ADC concentrated solutions containing target buffer salts.

[0047] (4) The protein concentrations of the ADC solutions after solution replacement and concentration were measured. The excipient concentrated solutions with corresponding amounts were added according to the calculated values, and the buffer salts were added to the target volume.

[0048] (5) The samples were subjected to filtration, subpackaging and then plugging and capping.Stability Investigation:

[0049] The samples were placed at 25° C. for a total of 2 months investigation. The investigation results are shown in Table 2, wherein TO represents the time point at which the samples began to be placed, 25° C.-1M represents the time point at which the samples were placed for one month, and 25° C.-2M represents the time point at which the samples were placed for two months.

[0050] TABLE 2Summary of stability investigation results for the formulations with different pH valuesProteinCEX-HPLC(%)AS269FormulationconcentrationpHMainAcidicBasicresiduenumberTime pointAppearance(mg / ml)valuepeakpeakpeak(%)FormulationT0Colorless and9.3535.45757.6528.1914.160.0181no visibleforeign matter25° C.-1 MColorless and10.2155.52851.4424.3624.210.036no visibleforeign matter25° C.-2 MColorless and10.0905.51342.1624.8732.970.028no visibleforeign matterFormulationT0Colorless and9.0645.72758.1328.713.17<0.0172no visibleforeign matter25° C.-1 MColorless and10.0065.80249.2627.6423.110.021no visibleforeign matter25° C.-2 MColorless and10.0015.74541.9329.3928.670.039no visibleforeign matterFormulationT0Colorless and9.3035.97659.0829.3311.58<0.0173no visibleforeign matter25° C.-1 MColorless and10.3336.07746.831.7021.49<0.015no visibleforeign matter25° C.-2 MColorless and10.3836.00536.5838.3625.050.023no visibleforeign matterFormulationT0Colorless and9.0316.3158.4429.8611.69<0.0164no visibleforeign matter25° C.-1 MColorless and10.1246.42841.2639.5519.18<0.015no visibleforeign matter25° C.-2 MColorless and10.0776.34430.8149.5819.60.018no visibleforeign matterFormulationT0Colorless and10.5157.18957.6729.1013.23<0.0165no visibleforeign matter25° C.-1 MColorless and10.0907.20130.9052.5016.60<0.016no visibleforeign matter25° C.-2 MColorless and9.9897.19422.4761.4919.05<0.016no visibleforeign matterFormulationT0Colorless and9.8723.98257.7126.9415.34<0.0166no visibleforeign matter25° C.-1 MColorless and10.3293.958NDNDND0.18no visibleforeign matter25° C.-2 MColorless and10.1923.988NDNDND0.234no visibleforeign matterFormulationT0Colorless and9.8044.53156.9327.5315.53<0.0167no visibleforeign matter25° C.-1 MColorless and10.3304.495NDNDND0.111no visibleforeign matter25° C.-2 MColorless and10.2114.526NDNDND0.154no visibleforeign matterFormulationT0Colorless and9.5055.0256.9127.2915.79<0.0168no visibleforeign matter25° C.-1 MColorless and9.9204.983NDNDND0.055no visibleforeign matter25° C.-2 MColorless and9.8785.031NDNDND0.083no visibleforeign matterFormulationT0Colorless and9.8465.53656.328.7114.98<0.0169no visibleforeign matter25° C.-1 MColorless and10.3665.51146.5322.9630.510.026no visibleforeign matter25° C.-2 MColorless and10.2595.5637.2823.6839.020.038no visibleforeign matterFormulationT0Colorless and9.9776.03757.5627.3315.1<0.01610no visibleforeign matter25° C.-1 MColorless and10.4606.03047.8828.9823.13<0.015no visibleforeign matter25° C.-2 MColorless and10.2256.09036.8535.3827.780.016no visibleforeign matterND: due to significant increase in AS269 residue at this time point, CEX was not tested.Result Analysis:

[0051] According to the AS269 residue results (Table 2), except for formulation 3 (pH 6.0), formulation 4 (pH 6.3) and formulation 10 (pH 6.0), the growth trend of AS269 residue is observed in all other formulations (pH<6.0) under the investigation at 25° C. The lower the pH is, the higher the AS269 residue is, indicating that the small molecule is prone to drop under the condition that pH is less than 6.0.

[0052] According to the CEX results, the decline of CEX main peak as well as the increase of acidic peak and basic peak are more likely to happen under the condition of pH>6.0.

[0053] It can be seen that the pH value of the freeze-dried composition is crucial for product stability. The pH value of the freeze-dried composition of the present invention is preferably between 5.7 and 6.3, more preferably around 6.0.Example 2 Formulation Stability Study Under Different ADC Concentrations and Polysorbate 80 Concentrations

[0054] According to the experimental results of Example 1, formulations 11-22 (Table 3) were designed to investigate the effect of different ADC concentrations and different polysorbate 80 concentrations on product stability. After placing the samples under the conditions of 25° C. and shaking at 200 rpm for 1 day and 3 days respectively, samples were taken for testing. The test results (Table 4) show that there are no significant changes in all indicators of the samples of the formulations 11-22 before and after shaking. Therefore, under the pH value of approximately 6.0, with the protein concentration of 5 mg / mL to 30 mg / ml and the polysorbate 80 concentration range of 0.01% to 0.05%, the protein stabilities are all good.

[0055] TABLE 3Formulations with different ADC concentrationsand polysorbate 80 concentrationsFormulationTrehaloseBufferpHAnti-HER2 ADCPolysorbate 80numberconcentrationsaltvalueconcentrationconcentrationFormulation 115%5 mM6.0 5 mg / ml0.01%Formulation 12histidine0.02%Formulation 13salt0.05%Formulation 1410 mg / ml0.01%Formulation 150.02%Formulation 160.05%Formulation 1720 mg / ml0.01%Formulation 180.02%Formulation 190.05%Formulation 2030 mg / ml0.01%Formulation 210.02%Formulation 220.05%

[0056] TABLE 4Summary of shaking stability results for the formulations withdifferent ADC concentrations and polysorbate 80 concentrationsProteinCEX-HPLC(%)AS269FormulationconcentrationpHMainAcidicBasicresiduenumberTime pointAppearance(mg / ml)valuepeakpeakpeak(%)FormulationT0Colourless and no5.206.00762.5027.1010.400.03111visible foreignmatterShaking forColorless and no5.006.03060.7028.6010.700.0311 dayvisible foreignmatterShaking forColorless and no5.425.98961.0028.0011.100.0313 daysvisible foreignmatterFormulationT0Colorless and no5.306.06562.3027.2010.500.03112visible foreignmatterShaking forColorless and no5.136.00360.0029.1010.900.0311 dayvisible foreignmatterShaking forColorless and no5.306.04360.4028.3011.200.0313 daysvisible foreignmatterFormulationT0Colorless and no5.305.98762.0027.3010.700.03113visible foreignmatterShaking forColorless and no5.226.06058.7030.2011.100.0311 dayvisible foreignmatterShaking forColorless and no5.066.00460.2028.5011.300.0313 daysvisible foreignmatterFormulationT0Colorless and no9.485.98761.9027.4010.700.01614visible foreignmatterShaking forColorless and no9.376.00061.9027.6010.500.0161 dayvisible foreignmatterShaking forColorless and no10.296.00260.7027.8011.500.0163 daysvisible foreignmatterFormulationT0Colorless and no9.395.98861.5027.8010.700.01615visible foreignmatterShaking forColorless and no9.346.02061.6028.0010.500.0161 dayvisible foreignmatterShaking forColorless and no9.825.96559.9028.3011.700.0163 daysvisible foreignmatterFormulationT0Colorless and no9.496.00762.0027.9010.100.01616visible foreignmatterShaking forColorless and no9.615.98860.7028.7010.600.0161 dayvisible foreignmatterShaking forColorless and no10.016.09061.1027.7011.200.0163 daysvisible foreignmatterFormulationT0Colorless and no21.005.98761.1027.7011.200.007817visible foreignmatterShaking forColorless and no20.615.98460.6027.9011.500.00781 dayvisible foreignmatterShaking forColorless and no20.956.00160.2027.2012.600.00783 daysvisible foreignmatterFormulationT0Colorless and no20.736.00260.9028.1011.000.007818visible foreignmatterShaking forColorless and no21.706.03460.3028.1011.600.00781 dayvisible foreignmatterShaking forColorless and no21.395.99361.6026.1012.300.00783 daysvisible foreignmatterFormulationT0Colorless and no20.996.09062.1027.4010.400.007819visible foreignmatterShaking forColorless and no20.826.00760.1028.9011.000.00781 dayvisible foreignmatterShaking forColorless and no21.205.98758.3029.8011.900.00783 daysvisible foreignmatterFormulationT0Colorless and no30.676.00260.2028.8010.900.005220visible foreignmatterShaking forColorless and no29.726.03056.6030.3013.100.00521 dayvisible foreignmatterShaking forColorless and no30.025.98860.0028.3011.700.00523 daysvisible foreignmatterFormulationT0Colorless and no31.276.00359.9029.1011.000.005221visible foreignmatterShaking forColorless and no29.956.04556.5030.3013.200.00521 dayvisible foreignmatterShaking forColorless and no31.066.01059.3029.0011.600.00523 daysvisible foreignmatterFormulationT0Colorless and no30.295.97860.6028.6010.700.005222visible foreignmatterShaking forColorless and no29.925.98056.8031.6011.500.00521 dayvisible foreignmatterShaking forColorless and no30.116.00259.4028.8011.800.00523 daysvisible foreignmatterExample 3 Study on Freeze-Drying Parameters of the Formulations with Different Solid Contents

[0057] Formulations 23-34 were designed to investigate the effect of different combinations of anti-HER2 ADC concentrations and trehalose concentrations on the freeze-drying parameter (Tg′ / Tc). The results of Table 5 show that the freeze-drying parameters of the formulations with different combinations of anti-HER2 ADC concentrations and trehalose concentrations exist differences. Considering the influence of the freeze-drying parameter (Tg′ / Tc), freeze-drying filling volume and anti-HER2 ADC concentration on freeze-drying appearance, process time and stability, the concentration of anti-HER2 ADC may be 10 mg / mL to 30 mg / ml and the concentration of trehalose may be 2.5% to 8%. Two formulations (formulation 23 and formulation 24), three formulations (formulation 27, formulation 28 and formulation 29) and one formulation (formulation 31) with anti-HER2 ADC concentration of 10 mg / ml, 20 mg / ml and 30 mg / ml respectively were taken to carry out freeze-drying. According to the results (Table 6), the freeze-dried product with formulation 28 has the best appearance performance.

[0058] TABLE 5Summary of freeze-drying parameter results for the formulations with different solid contentsGlass-PolysorbateAnti-HER2CollapsingtransitionFormulationBufferpH80TrehaloseADCSolidtemperaturetemperatureNumbersaltvalueconcentrationconcentrationconcentrationcontent(Tc)(Tg′)Formulation5 mM6.00.02%4.5% 10 mg / ml5.5% −24.0−28.5623histidineFormulationsalt7% 8%−24.5−30.3124Formulation9%10%−25.0−31.2125Formulation11% 12%−26.0−31.0826Formulation3.5% 20 mg / ml5.5% −20.0−25.2427Formulation6% 8%−22.5−26.1128Formulation8%10%−22.5−26.9929Formulation10% 12%−24.3−26.5230Formulation2.5% 30 mg / ml5.5% −15.6−24.5931Formulation5% 8%−17.7−25.5032Formulation7%10%−21.0−25.9433Formulation9%12%−19.0−26.1134

[0059] TABLE 6Summary of freeze-drying appearances for different formulationsFreeze-drying processMoisturePre-PrimarySecondarycontentFormulationfreezingsublimationsublimationAppearance(%)FormulationPrefreezingHeating upHeating upTop cracking within 2 vials, top cracking1.52023at −45° C.to −30° C. atto 25° C. atand bottom shrinkage within 10 vials,for 5 hours5° C. / h and11° C. / h andbottom collapse within 1 vial andholding forholding forcomplete melting within 1 vial.Formulation40 hours3 hours withTop cracking and bottom shrinkage2.55724with aa vacuumwithin 11 vials, and bottom collapsevacuumdegree of 12within 3 vials.Formulationdegree of 12PaSurface cracking within 19 vials, and0.87827Paside collapse within 1 vial.FormulationSlight top cracking, slight bottom0.91828cracking and detachment within 20 vials.FormulationSurface cracking and detachment within0.8892916 vials, and bottom shrinkage anddetachment within 4 vials.FormulationDetachment and fragmentation within 290.87531vials.Example 4 Freeze-Drying Process Study

[0060] Formulation 28 of Example 3, that is, 20 mg / mL of anti-HER2 ADC (ARX788), 5 mM of histidine, 6 wt. % of trehalose and 0.02 wt. % of polysorbate 80, was used to investigate the freeze-drying process.

[0061] ADC samples and placebo (5 mM of histidine, 6 wt. % of trehalose and 0.02 wt. % of polysorbate 80) were prepared by referring to the sample preparation steps (1)-(4) in Example 1. The results are shown in Table 7. From the results of Table 7, it can be seen that the temperatures, vacuum degrees and other process parameters of primary sublimation and secondary sublimation will affect the appearance, moisture content and other properties of the obtained freeze-dried products. The appearances of the freeze-dried products obtained from process 1′ are severely unqualified, and the moisture content is high, which is not conducive to preparation stability.

[0062] Although the appearances of the freeze-dried products obtained from process 1 have been significantly improved, there are still a certain proportion of unqualified products. For processes 2-4, the processes of primary and secondary sublimations are comprehensively considered and optimized, resulting in the freeze-dried products with excellent appearances and low moisture content, thus the yield and product stability of the freeze-dried preparation can be significantly improved.

[0063] TABLE 7Freeze-drying process studyBatchProcess-1′Process-1Process-2Process-3Process-4Shelf area of freeze-dryer5m20.2m20.2m20.2m25m2Sample description123 vials of ADC,108 vials of5 vials of ADC, with20 vials of ADC, with27 vials of ADC, withwith the rest filledplacebosthe rest filled withthe rest filled withthe rest filled withwith placebosplacebosplacebosplacebos(2 shelves)(2 shelves)(2 shelves)(2 shelves)Pre-freezing methodCooling down the shelf to 4° C. within 1 hour and holding at the same temperature for 1 hour, thencooling down the shelf to −45° C. within 3.5 hours and holding at the same temperature for 5 hoursPrimaryShelf temperature−30°C.−30°C.−20°C.−23°C.−23°C.sublimationHeating up time7.5h0.5h0.5h0.5h0.5hHolding time40 hVacuum degree22Pa13Pa13Pa13Pa13PaSecondaryShelf temperature25°C.25°C.30°C.25°C.25°C.sublimationHeating up time 5 hHolding time5h8h8h8h8hVacuum degree22Pa13Pa13Pa13Pa13PaTestAppearanceThere are aboutThere is slightThere is slightThere is slightThere is no shrinkageresult90% of ADCshrinkage at theshrinkage at the edge.shrinkage at the edge.or layering in thesamples fail visualbottom of theThere are slight cracksThere are slight cracksmiddle of the druginspection, withvial. 5 out ofat the bottom of theat the bottom of thecake in the ADCpartial shrinkage at108 vials areADC and no cracks atADC and no cracks atsamples, part ofthe bottom of theunqualified inthe bottom of thethe bottom of thewhich have cracks.vial wall andappearance.placebo. All of themplacebo. All of themAll of them arelayering in theare qualified inare qualified inqualified inmiddle of the drugappearance.appearance.appearance.cake.Moisture content1.738% (ADC)0.667% (placebo)0.545% (ADC)0.469% (ADC)0.717% (ADC)1.823% (placebo)0.533% (placebo)0.737% (placebo)0.898% (placebo)pH5.9966.0345.9896.0126.019ADC30s41s43s34s21sreconstitution timeCEXMain57.17%57.24%57.67%58.09%57.63%peakAcidic26.39%26.30%25.89%25.39%25.52%peakBasic16.44%16.46%16.44%16.52%16.85%peakSECMain95.90%95.47%96.00%96.13%95.72%peakHMW4.10%4.53%4.00%3.87%4.28%LMWNot detectedNot detectedNot detectedNot detectedNot detectedAS269 residue0.017%0.016%0.016%0.016%0.015%Example 5 Pilot Scale Production and Stability Investigation

[0064] Three batches of pilot scale production were carried out according to process 4 determined in Example 4 (i.e., pilot scale batch −1 to pilot scale batch −3, with a batch size of approximately 1500 vials). The process was as follows:

[0065] (1) ADC drug substance was taken from the refrigerator 24 hours in advance and placed at room temperature for thawing. After the thawing was completed, the drug substance was merged into a Schott bottle, and stirred and mixed thoroughly.

[0066] (2) The mixed drug substance was sterilized and filtrated through a two-stage 0.22 μM sterile filter into a sterile storage bag.

[0067] (3) Penicillin vials were sterilized through a tunnel drying oven and delivered to an isolator, and rubber stoppers and aluminum covers were delivered to the isolator through an RTP bucket after steam sterilization.

[0068] (4) The sterile storage bag was connected to the isolator through a sterile connection, then the solution was filled into the sterilized penicillin vials.

[0069] (5) The penicillin vials were semi-stoppered, and then transferred to a freeze-dryer, in which the shelves needed to be all filled, and the vacancy needed to be filled with empty penicillin vials. The program for freeze-drying was started, and after the freeze-drying was completed, the vials were full-stoppered.

[0070] (6) After being full-stoppered, the penicillin vials were taken out of the freeze-dryer and delivered to a capping area for capping.

[0071] (7) After capping, the penicillin vials were subjected to outer surface cleaning, visual examination, labelling and packaging.

[0072] Production data shows that all three batches of the production are successfully completed, and the freeze-dried products are white or off-white blocks in appearance. The yield of finished products is high, and there are no defective products like remelting or collapse. All the reconstitution time is less than 1 minute, and all the moisture contents are less than or equal to 1%.

[0073] For the sterile powder for injection produced by the method described in this example, the stability of the three batches of samples under long-term (5±3° C.) conditions for 24 months and accelerated (25±2° C.) conditions for 6 months was evaluated by using appearance, pH value, reconstitution time, moisture content, protein content, drug-antibody ratio, SEC, CEX, CE-SDS, AS269 residue, cell-based potency, binding potency, etc. as indicators. The results show that there are no significant changes in all indicators before and after the evaluation.

[0074] Meanwhile, one batch of the samples (pilot scale batch −2) was subjected to high-temperature impact factor investigation. The results show that the samples have very good stability, even after a 6-month evaluation at 40° C., the change ranges of CEX peak (FIG. 2A) and potency (FIG. 2B) both are very small.

[0075] Unless otherwise particularly limited, the terms used in the present invention are commonly used meanings understood by those skilled in the art.

[0076] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes, and improvements within the scope of the present invention. Therefore, the present invention is not limited to the aforementioned embodiments but is only limited by the claims.

Claims

1. A freeze-dried composition containing anti-HER2 drug conjugate, wherein the freeze-dried composition comprises 10 mg / mL to 30 mg / mL of ARX788, 5 mM to 10 mM of histidine, 2.5 wt. % to 8 wt. % trehalose and 0.01 wt. % to 0.05 wt. % of polysorbate 80, wherein the pH value of the freeze-dried composition is from 6.0 to 6.3.

2. A freeze-dried preparation containing anti-HER2 drug conjugate, wherein the freeze-dried preparation is prepared from the freeze-dried composition according to claim 1.

3. A preparation method for a freeze-dried preparation of the freeze-dried composition according to claim 1, comprising the following steps:S1: prefreezing the freeze-dried composition at −50° C. to −40° C. for 3 hours to 6 hours;S2: heating up the freeze-dried composition after treated in step S1 to −25° C. to −20° C. and holding at this temperature and a vacuum degree of 10 Pa to 15 Pa for 30 hours to 50 hours; andS3: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. and holding at this temperature and a vacuum degree of 10 Pa to 15 Pa for 5 hours to 10 hours.

4. The preparation method according to claim 3, wherein the step S1 comprises: cooling down the freeze-dried composition to 2° C. to 5° C. within 0.5 hour to 1.5 hours and holding at the same temperature for 0.5 hour to 1.5 hours, then cooling down to −50° C. to −40° C. within 3 hours to 5 hours and holding for 3 hours to 6 hours at the same temperature.

5. The preparation method according to claim 3, wherein the step S2 comprises: heating up the freeze-dried composition after treated in step S1 to −23° C. to −20° C. within 0.2 hour to 0.8 hour and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 35 hours to 45 hours.

6. The preparation method according to claim 3, wherein the step S3 comprises: heating up the freeze-dried composition after treated in step S2 to 25° C. to 30° C. within 2 hours to 8 hours and holding at the same temperature and a vacuum degree of 12 Pa to 14 Pa for 5 hours to 10 hours.

7. A method for treating a cancer by administering the freeze-dried composition according to claim 1 or a freeze-dried preparation thereof, to a subject in need, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, urothelial cancer, biliary tract cancer, liver cancer, brain cancer, esophageal cancer, endometrial cancer, uterine cancer, kidney cancer, bladder cancer, thyroid cancer, salivary gland cancer or pancreatic cancer.

8. The freeze-dried composition according to claim 1, wherein the pH value of the freeze-dried composition is 6.0.

9. The freeze-dried composition according to claim 8, wherein the freeze-dried composition comprises 20 mg / mL of ARX788, 5 mM of histidine, 6 wt. % trehalose and 0.02 wt. % of polysorbate 80.

10. A freeze-dried preparation containing anti-HER2 drug conjugate, wherein the freeze-dried preparation is prepared from the freeze-dried composition according to claim 9.

11. A method for treating a cancer by administering the freeze-dried composition of claim 8 or the freeze-dried preparation thereof, to a subject in need, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, urothelial cancer, biliary tract cancer, liver cancer, brain cancer, esophageal cancer, endometrial cancer, uterine cancer, kidney cancer, bladder cancer, thyroid cancer, salivary gland cancer or pancreatic cancer.

12. A method for treating a cancer by administering the freeze-dried composition of claim 9 or the freeze-dried preparation thereof, to a subject in need, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, urothelial cancer, biliary tract cancer, liver cancer, brain cancer, esophageal cancer, endometrial cancer, uterine cancer, kidney cancer, bladder cancer, thyroid cancer, salivary gland cancer or pancreatic cancer.

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